Part:BBa_K1813020

nicC Expression Cassette with LacI Reversed

LacI Reversed nicC

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 105
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1658
  
• 1000
  COMPATIBLE WITH RFC[1000]

Background of LacI Ptac NicC Term

Part Description:

6-chloronicotinic acid (6-CNA) is an intermediate in imidacloprid degradation that is both toxic to bees (1), and a persistent environmental contaminant (2).The conversion from 6-CNA to 6-HNA, a well studied intermediate in nicotine degradation (3), is catalyzed by 6-chloronicotinic acid chlorohydrolase (cch2), a chlorohydrolase from SG-6C Bradyrhizobiaceae (4).

Design and Acquisition

After synthesizing a codon-optimized nicC, we used standard assembly to created a composite part composed of nicC driven by the Ptac promoter (BBa_K1813037) and flanked by a double terminator (BBa_B0014). The tac promoter contains a lac operator sequence that can be bound by LacI, the lac repressor protein, allowing inducible expression by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Our nicC expression cassette was assembled behind a lacI cassette (BBa_K1813019) to give us the ability to control the expression of nicC. All nicC constructs are contained within standard pSB1C3 vectors.

Experience

SDS PAGE Protein Expression for nicC

nicC was expressed in E.Coli DH5-α. Transformed E.Coli was grown at 37°C until an OD600 of 0.6 to 0.8. They were then induced with IPTG and grown overnight at 16°C, 20°C, 25°C, 30°C, 37°C to discern which temperature resulted in optimal protein expression. The samples were prepared for SDS page gel via the SDS page sample preparation protocol and SDS page gel protocol (reference).

nicC has over-expression at 25°C and 30°C at the expected size of 52kDa, however there is a significant amount of insoluble fraction of the gene product.

Figure 1: 12% SDS-PAGE gel run with nicC. No discernible expression is seen at the expected size of nicC, 43kDa.